Effect of isoacteoside on cell viability
The wide diversity of natural products found in plants enables the discovery of novel compounds to combat chronic diseases such as those caused by obesity. Isoacteoside, a phenylethanoid glycoside and an isomer of acteoside (Fig. 1), has been isolated from a number of plant species, including Magnolia denudata, where it is found in the flower [3]. A few studies have demonstrated the physiological activity of isoacteoside. Isoacteoside inhibits a-amylase and lipase [7] and inhibits the production of pro-inflammatory cytokines in the human mast cell line HMC-1, by blocking the caspase‑1, MAPK, and NF-kB signaling pathways [8]. Isoacteoside attenuates acute kidney injury induced by severe acute pancreatitis and septic acute lung injury. In addition, it exerts antioxidant, anti-inflammatory, and anticancer effects [9,10,11]. However, the in vitro bioactivity of isoacteoside with regard to its anti-obesity effects has not been studied. In this study, the anti-obesity effects of isoacteoside were investigated in 3T3-L1 pre-adipocytes.
The effects of isoacteoside on the viability of 3T3-L1 cells are shown in Fig. 2. Despite increasing the concentration of isoacteoside, cell viability remained unchanged until a certain concentration was reached. After 24 h of isoacteoside treatment, cell viability was approximately 100.67 ± 0.15, 102.64 ± 0.31, 103.42 ± 0.58, 100.88 ± 0.17, 98.57 ± 0.93, 28.78 ± 2.77 and 7.82 ± 0.19% in comparison to the control group for 100, 300, 500, 1000, 1500, 2000, and 2500 μM isoacteoside-treatment groups, respectively. After 48 h of isoacteoside treatment, cell viability was approximately 104.51 ± 4.25, 100.80 ± 9.11, 104.80 ± 5.48, 106.10 ± 7.33, 48.41 ± 3.67, 3.10 ± 0.07, 3.09 ± 0.02% in comparison to the control group for 100, 300, 500, 1000, 1500, 2000, and 2500 μM isoacteoside-treatment groups, respectively. After 72 h of isoacteoside treatment, cell viability was similar to 48 h (data was not shown). These data suggest that isoacteoside does not significantly affect cell viability below a concentration of 1000 μM. Therefore, we inferred that while isoacteoside has almost no effect on cell viability, it might have other effects, such as on the adipogenic differentiation of 3T3-L1 cells.
Effect of isoacteoside on differentiation of 3T3-L1 cells
The pre-adipocytes were treated with differentiation medium, containing DMEM with IBMX, DEX, and INS in order to generate lipid droplets. These lipid droplets developed a red color upon staining with oil red O, as observed from the cell culture plates (Fig. 3A) and by optical microscopy (Fig. 3B). However, although the cells were treated with differentiation medium, lipid concentration decreased from 100% (control without treatment) to 68.69 ± 1.86% and 33.85 ± 0.22%, following treatment 75 and 150 mM isoacteoside, respectively. This result indicates that with an increase in the concentration of isoacteoside, the lipid content decreases (Fig. 3C). These results suggest that isoacteoside inhibits the differentiation of 3T3-L1 cells and thus the formation of lipid droplets.
Effect of isoacteoside on the expression of mRNAs related to adipogenesis and lipogenesis
To observe the extent of inhibition of adipogenesis and lipogenesis by isoacteoside, qRT-PCR was performed to quantify the expression levels of the related genes (Fig. 4). The primer sequences used for this experiment are listed in Table 1. The mRNA expression of peroxisome proliferator-activated acceptor gamma (PPARγ) was 10.78 ± 2.62% in the undifferentiated control group (− / −), compared to that in the differentiated control group (+ / −). The mRNA expression of PPARγ decreased to 42.34 ± 1.08% and 13.13 ± 0.17%, following treatment with 75 μM and 150 μM isoacteoside (Fig. 4A), respectively. The mRNA expression of CCAAT/enhancer-binding protein alpha (C/EBPα) was 2.65 ± 0.17% in the undifferentiated control group (− / −), compared to that in the differentiated control group (+ / −). The mRNA expression of C/EBPα decreased to 46.15 ± 0.70% and 8.73 ± 1.09% upon treatment with 75 μM and 150 μM isoacteoside, respectively (Fig. 4B). The mRNA expression of perilipin (PLIN1) was 0.63 ± 0.06% in the undifferentiated control group (− / −), compared to that in the differentiated control group (+ / −). The mRNA expression of PLIN1 decreased to 45.25 ± 2.53% and 6.13 ± 1.58%, upon treatment with 75 μM and 150 μM isoacteoside, respectively (Fig. 4C). These results suggest that isoacteoside suppresses the expression of genes related to adipogenesis and lipogenesis.
Effect of isoacteoside on the expression of adipogenesis and lipogenesis-related proteins
The expression of three proteins involved in the adipogenesis of 3T3-L1 cells and lipid droplet accumulation is shown in Fig. 5A. The expression of PPARγ in the undifferentiated control group (− / −) was 17.16 ± 3.85% compared to that in the differentiated control group (+ / −). PPARγ expression levels were 38.50 ± 3.14% and 30.02 ± 8.42% in the groups treated with 75 μM and 150 μM isoacteoside, respectively, compared to that in the differentiated control group (+ / −) (Fig. 5B). The expression of C/EBPα in the undifferentiated control group (− / −) was 12.53 ± 1.72% compared to that in the differentiated control group (+ / −). C/EBPα expression levels were 33.16 ± 3.42% and 18.81 ± 0.22% in the groups treated with 75 μM and 150 μM isoacteoside, respectively, compared to that in the differentiated control group (+ / −) (Fig. 5C). The expression of PLIN1 in the undifferentiated control group (− / −) was 8.22 ± 1.55% compared to that in the differentiated control group (+ / −). PLIN1 expression levels were 31.70 ± 1.79% and 6.35 ± 2.78% in the groups treated with 75 μM and 150 μM isoacteoside, respectively, compared to that in the differentiated control group (+ / −) (Fig. 5D).
PPARγ and C/EBPα are two important proteins involved in adipogenesis and lipogenesis, respectively [12]. PPARγ is a nuclear receptor protein within the PPAR group and acts as a transcription factor involved in the differentiation of pre-adipocytes and lipid metabolism [13]. PPARγ targets fat-specific marker genes such as genes encoding adipocyte fat acid-binding protein (aP2) and sterol regulatory element-binding protein 1 (SREBP-1). PPARγ promotes adipose tissue production and reduces the expression of leptin, thereby inhibiting lipolysis and promoting lipid accumulation [14,15,16]. C/EBPα is also a transcription factor that binds aP2 with PPARγ and regulates adipogenesis [17, 18]. C/EBPα is PPARγ-dependent, indicating that C/EBPα alone cannot induce adipogenesis in the absence of PPARγ [19]. Perilipin is a protein found in lipid droplets present on the surface of differentiated adipocytes, where it blocks access to lipases to regulate lipogenesis and promotes PPARγ expression [20]. Therefore, our results indicate that the expression of adipogenesis and lipogenesis-related proteins was suppressed by isoacteoside treatment (Fig. 5). In general, natural products have few side effects on the human body, except in a few cases [21].
The present study aimed to investigate whether isoacteoside inhibits adipogenesis and lipogenesis in 3T3-L1 pre-adipocytes. Although the viability of 3T3-L1 cells was not affected by isoacteoside (< 1000 µM), anti-obesity effects were observed at concentrations lower than 1000 µM. This was supported by the fact that lipid content decreased in the cell culture after treatment with 75 μM and 150 μM isoacteoside, as shown by oil red O staining and the expression levels of adipogenesis- and lipogenesis-related proteins and mRNAs. These results showed that isoacteoside exerted anti-obesity effects on 3T3-L1 cells.
Although, more studies are needed to support this promising mechanism, this study might provide implication in animal and human anti-obesity effect. However, isoacteoside will need to be compared for their efficacy with lipid-lowering medications, such as simvastatin, mevinolin, or orlistat. In the future, in vivo studies analyzing other tissues involved in obesity are required to validate the anti-obesity effects of isoacteoside.